The introduction of D-amino acid residues into peptide hormones has been traditionally utilized in structure-activity studies to probe the conformational requirements of ligand-receptor interactions. A study was undertaken to examine the effect of D-amino acid substitutions into the atrial natriuretic peptide molecule on interactions with distinct subpopulations of specific membrane-associated receptors of bovine aortic smooth muscle cells. Competitive binding analysis revealed that each of 15 synthetic D-amino acid-substituted analogs showed comparable affinities for C-ANP receptors, a class of specific receptors which have been proposed to mediate the sequestration and metabolic clearance of ANP. The relative affinities of all 15 analogs did not differ more than 10-fold. In contrast, the interaction of the ANP analogs with a second receptor pool (B-ANP receptors), which is coupled to the stimulation of particulate guanylate cyclase, varied over a 1000-fold range of potency consistent with expectations for a receptor that displays rigorous conformational specificity. The indiscriminant selectivity of C-ANP receptors for D-amino acid-substituted ANP analogs is unprecedented for hormone receptors involved in biological signal transduction. These results, when coupled with the inability to correlate any direct in vitro biological effect associated with C-ANP receptor occupancy supports the hypothesis that the C-ANP receptor protein is a novel transport protein involved in the metabolic clearance of ANP.